Maprotiline Prompt the Anti-tumor Effect by Inhibiting the PD-L1 in Mice Burdened Melanoma

Tiesuo Zhao Xinxiang Medical University Yang Li Xinxiang Medical University Miaomiao Liu Xinxiang Medical University Lin Zhou Xinxiang Medical University Zunge Wu Xinxiang Medical University Haoqi Chen Xinxiang Medical University Jiaming Guo Xinxiang Medical University Mengdan Gao Xinxiang Medical University Jiateng Zhong Xinxiang Medical University Mingyong Wang Xinxiang Medical University Feng Ren Xinxiang Medical University Zhiwei Feng Xinxiang Medical University Huijie Jia (  zhongziqi1115@163.com ) Xinxiang Medical University https://orcid.org/0000-0002-4267-0198


Introduction
Melanoma is one of the most common fatal malignancies in the world. In the past, the outcomes of patients have been improved dramatically, but the overall survival rates with 5-year is only 30% to 40% [1].
Most patients don't achieve the therapeutic effect as expected. It is still necessary to study new drug for the treatment of melanoma.
It is considered an effective and safe route to nd novel indications of old drug. As known, antidepressants have been widely used for the treatment of a variety of diseases including depressive states and mood in clinical [2]. Though antidepressants are found that may promote tumor growth and it is correlating with dose dependent in some studies [3,4], other studies showed the con ict results that antidepressants could play the anti-tumor effect [5,6] and did not revealed the disadvantageous risk in tumor patients [7,8]. Thus, the new functions of antidepressant to treat tumors still need to discuss stirringly in the future.
Maprotiline, as a tetracyclic antidepressant, has been widely used for treating mental depression in clinical [9,10]. With the development of studies, the most antidepressants have been demonstrated that could play an anti-tumor role [11,12]. Of course, maprotiline also is been proved that have potent antiproliferative effect on Burkitt lymphoma [13]. In addition, a study found that maprotiline has a dual effect on autophagy of Neuro-2a cells. It not only induces apoptosis through the caspase-3 pathways, but also causes an anti-apoptotic response through the both pathways of Ca2 + -and ERK-dependent [2]. Thus, it still needs to be further studied on the anti-tumor mechanism of maprotiline.
With the development of immunology, tumor immunotherapy is getting more and more attention. Checkpoint immunotherapy, as a strategy of tumor immunotherapies, has been demonstrated that could induce clinical bene t on treatment tumors by targeting the inhibitory co-receptors such as PD-1 [14,15]. PD-1 combination with PD-L1, which is expressed on the surface of tumor cells, inhibits the anti-tumor function of T cells. It has been proved that PD-L1 overexpresses on the melanoma [16]. Inhibited of the pathway of PD-1/PD-L1 could attenuate melanoma growth by strengthening the anti-tumor effect of T cells [17]. However, it is not clear whether maprotiline in uences the expression of PD-L1 on the melanoma and the immune response against tumor.

Cell lines and animals
The B16 cells were presented by Professor Wang Liying (Department of molecular biology, Jilin University). C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were fed with food and water in sterile condition.
Wound healing assay B16 cells were seeded into the 6-well plate with the density of 3.5 × 10 5 cells/well and incubated in an incubator (37℃, 5% CO 2 ) for 14-16h. Then the cells were scratched a hole in the well center with the tip of the 100 μl pipette and cultured for 24 hours or 48 hours with different concentrations of maprotiline (0, 0.625, 1.25, 2.5, 5 and 10 μg/ml). The scoring of cells in each well was recorded using the microscope.
Cell Counting Kit-8 analysis B16 cells were prepared into 96-well plates with a density of 1.5×10 4 cells/well and incubated for 14-16h at 37℃ with the condition of 5% CO 2 . Then maprotiline with the different concentrations of 0, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20 and 40μg/ml were added, respectively. After co-cultured for another 24 hours or 48 hours, CCK-8 was added to each well and incubated for 2 hours according to the instruction. Finally, the OD value at the wavelength of 450nm was detected by an enzyme-labeling instrument (Molecular Devices, USA). The cells in each well were photographed under the microscope.
Annexin V/PI analysis B16 cells were seeded into the 6-well plate with the density of 3.5×10 5 cells/well and cultured in an incubator (37 ℃, 5% CO 2 ) for 14-16h. The maprotiline with the different concentrations of 0, 0.625, 1.25, 2.5, 5 and 10μg/ml was respectively added into the well. After 24 hours or 48 hours, the cells were collected and mixed with 100μl Annexin V binding buffer. Then the cells were labeled with FITC and PI antibodies. The ratio of apoptosis was analyzed by the ow cytometry (Cyto ex, Beckman).

Establishment of animal models and treatment
Female C57BL/6 was used for establishing animal model of tumor burdened. The B16 cells at the dose of 1×10 6 per mouse were injected subcutaneously in the right leg of mice to establish the melanoma bearing mice model. At 7 days after tumor inoculation, mice were randomly divided into PBS group, maprotiline 100μg group and maprotiline 200μg group. After the treatment, the survival rate of mice was recorded every day. At 14 days after the treatment, the tumor was separated and weighed. The spleens also were collected for detecting the ratios of immune cells using the ow cytometry.

Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nickend labeling (TUNEL)
The apoptosis of cells in tumor-tissues is detected by TUNEL assay kit (Beyotime Institute of Biotechnology, Shanghai, China) according the instruction. Firstly, the TUNEL detection solution is dropwise added on the surface of tumor sections and incubated in dark at 37℃ for 60min. Then the sections are washed using PBS for 10min and 3 times. Finally, the sections are dried and sealed with the solution of anti-uorescence quenching, and observed the image using the uorescence microscope.

Immuno uorescence staining
The tumor sections were incubated with monoclonal antibodies of CD3 (1:100) CD4 (1:150) CD8 (1:600), (Cell Signaling Technology, USA) overnight at 4 ℃. The sections were rinsed by PBS for three times and then incubated with the second antibody for 30min at room temperature. Finally, the sections were sealed with anti-uorescence quenching and observed under confocal microscope (AR1 + , Nikon).

Flow cytometry
The spleens were grinded and ltered through a nylon cell strainer lter, and then collected in PBS buffer. The Red Blood Cells (RBCs) were removed with Red Blood Cell Lysis Buffer (Beyotime Biotechnology, China). The spleen cells were resuspended in PBS buffer and adjust the concentration to 1×10 7 cells/ml.

Statistical analysis
Data are designated as the means ± SDs. One-way ANOVA was performed to test if mean values between different groups were signi cantly different (P <0.05). For survival analysis, the Kaplan-Meier method with a log-rank test was used.

Results
Maprotiline inhibited the proliferation of melanoma cells Firstly, we tested the viability of maprotiline with different concentrations on melanoma B16 cells by CCK8 assay. The results showed that maprotiline at the concentration of 20μg/ml or 40μg/ml could effectively inhibit the proliferation of B16 cells at 24h after treatment with maprotiline, while at 48h after treatment, the cell proliferation was effectively inhibited at the concentration of 40μg/ml, indicating that maprotiline could effectively inhibit the proliferation of B16 cells (Fig. 1).

Maprotiline inhibited the migration of melanoma cells
The effects of maprotiline with different concentrations on the migration of B16 cells were detected by wound healing assay. The results showed that maprotiline could effectively inhibit cell migration when the concentration of maprotiline was greater than 2.5μg/ml for 24h after the action on B16 cells, and signi cantly inhibited when the concentration of maprotiline was more than 5μg/ml for 48h, indicating that maprotiline has the ability to inhibit the migration of B16 cells (Fig. 2).

Effect of maprotiline on autophagy, apoptosis and migrate of B16 cells in vivo
Then, we used Western blotting to detect the effect maprotiline on the expression of tumor related proteins in B16 cells. We found that maprotiline increased the expression of autophagy related protein LC3b and apoptosis related protein cleaved caspase 3 in B16 cells when the concentration was more than 2.5μg/ml for 24 hours, while maprotiline with the concentration of 10μg/ml inhibited the expression of migration related protein MMP2. When the concentration of maprotiline was more than 5μg/ml, the expression of LC3b and cleaved caspase-3 were increased signi cantly, but there was no signi cant effect on the expression of MMP2 ( Fig. 3a and b). These results indicated that maprotiline did not inhibit B16 migration through MMP2 pathway. Subsequently, ow cytometry was used to detect the effect of maprotiline at different concentration on B16 cell apoptosis. The results showed that after treated for 24h or 48h, the concentration of maprotiline greater than 5μg/ml signi cantly increased B16 cell apoptosis, especially when the concentration was 10μg /ml ( Fig. 3c and d).
Effect of Maprotiline on the mice burdened the B16 cells Next, we further examined the therapeutic effect of maprotiline on melanoma tumor-bearing mice in vivo. The results showed that maprotiline with the concentration of 100μg/mouse and 200μg/mouse could effectively inhibit tumor growth in melanoma bearing mice and signi cantly prolonged the survival rate, but there was no signi cant difference between the two treatment groups (Fig. 4a-d).
Meanwhile, in order to further clarify the anti-melanoma effect of Maprotiline, we further examined the effect of Maprotiline on the expression of related proteins in tumor tissues. According to the results, maprotiline with the concentration of 100μg/mouse or/and 200μg/mouse signi cantly increased the expression of apoptosis related protein Cleaved-Caspase3 (Fig. 4e) which was coincident with the TUNEL results (Fig. 4f), but there is no signi cant effect on the expression of LC3b. This is not consistent with that of the results of cell experiments, which might be related to the dose of the drug, or may also be related to the in ltration of a large number of proliferating T lymphocytes in tumor tissues, affecting the expression of autophagy-related proteins. In both vitro and vivo, maprotiline has no obvious effect on the expression of MMP2. Importantly, we found that maprotiline inhibited the expression of PD-L1 in tumor tissues (Fig. 4e).
Effect of maprotiline on the ratio of CD4 + , CD8 + T lymphocytes and NK cells in spleen As the largest peripheral immune organ in the body, the spleen is the main site of immune response. We measured the ratio of CD4 + and CD8 + T lymphocytes in the spleen. The results showed that compared with the PBS group, the rate of in ltration of CD4 + and CD8 + T lymphocytes in the spleen were signi cantly increased in the two maprotiline treatment groups. However, compared with the low-dose group, the proportion of T lymphocytes in the spleen of the high-dose group was signi cantly reduced ( Fig. 5a and b). This may be related to the in ltration of numerous T lymphocytes into the tissue. In addition, we found that both treatment groups signi cantly increased the number of NK cells in the spleen compared with the PBS group, and there was no signi cant difference between the two groups ( Fig. 5c).

Effect of maprotiline on the in ltration of T lymphocytes in tumor tissues
To further determine whether maprotiline enhanced the anti-tumor immune response in melanoma bearing mice, we used immuno uorescence techniques to detect the in ltration of CD4 + and CD8 + T lymphocytes in tumor tissues. The results showed that, compared with the PBS group, maprotiline signi cantly increased the in ltration of CD4 + and CD8 + T lymphocytes in the tissues. However, the in ltration of T lymphocytes in tumor tissue was more pronounced in the high-dose group (200μg/mouse) than in the low-dose group (100μg/mouse) (Fig. 6). This may be related to the apoptosis of mass tumor cells, which leads to the release of more antigens, thus chemotactic more T cells to the tumor tissue.

Discussion
Searching the safe and effective drugs for tumor treatment is the focus of research, but it is expensive and time consuming to develop of novel drugs. At present, some old drugs of non-antitumor have been found that could play the effect of anti-tumor in some studies [18][19][20][21]. Because these drugs have been successfully used to treat of non-tumor diseases in clinical, it is helpful to develop therapeutic drugs for cancer patients. Maprotiline, an antidepressant drug, has been found that could decrease proliferation of prostate cancer cells [22], but the anti-tumor mechanism in melanoma are needed to evaluate. Here, we nd that maprotiline has a potent anti-tumor effect through prompting the immune response.
As known, the immune response of anti-tumor is impaired in patients, and the pathway of PD-1 and PD-L1 is an important cause of immune escape for tumor cells [23]. At present, the checkpoint inhibitors of PD-1 and PD-L1 have been approved or investigated for treatment tumor by FDA [24]. In this study, we con rm that maprotiline inhibited the growth of melanoma in mice burdened B16 cells, prolong the survival of mice. In order to explore the anti-tumor mechanism, we detect the expression of related protein and found that maprotiline inhibits the expression of PD-L1. Blocking the link of PD-1 and PD-L1 could recover the T cell function to destroy tumor cells [25]. It is might a reason that maprotiline delays the growth of melanoma. In addition, blocking the pathway of PD-1/PD-L1 could lead to signi cantly increase the ratios of T cells in spleen [26]. Our results also con rm that maprotiline increases the percentage of CD4 + and CD8 + T cells in the spleen. However, we nd that the ratios of T cell are higher in mice treated with maprotiline at the dose of 100μg per mouse compared with maprotiline at the dose of 200μg per mouse. It might relate with that more T cells in ltrate in the tumor tissues.
In this study, we nd that maprotiline signi cantly increase the T cell in ltration in tumor, and the degree of T cell in ltration is most remarkable in mice treated maprotiline at the dose of 200μg per mouse.

Though Liu et al indicates that expression of PD-1 only in uences the density of CD8 + T cell in ltration in
tumor tissue, not correlates with CD4 + T cells [27], but other study points out that inhibition the bind of PD-1 and PD-L1 not only increases the percentage of T cells in the spleen, but also increases the number of T cells in ltration in tumor tissue [26]. We analyze that maprotiline causes the apoptosis of copious tumor cells [2] which could produce of more tumor antigens recognized by antigen presentation cells (APC). Finally, the APCs could further lead to T cell activation [28] and in ltration in tumor-tissues. In fact, maprotiline increases the apoptosis of B16 cells and the expression of cleaved-Caspase3. In addition, accumulating evidence suggests that chemotherapy could induce immunogenic cell death (ICD) and prompt the anti-tumor e cacy [28]. It also might be a reason that causes the strengthened T cell response. Of course, we convince that effect of maprotiline on T cell in ltration might be complex and need to be further explored.
Besides in uencing the ratios of T cells, we also found that maprotiline increases the ratio of natural killer (NK) cells in the spleen. As known, NK cells belong to the innate lymphocytes that could destroy the tumor cells without MHC restricted manner [29]. However, the function of NK cells are suppressed by PD-L1 expressed on the surface of tumor cells [30]. Treatment with PD-1 monoclonal antibody could strengthen the NK cell function inhibited by PD-L1-mediated. Therefore, we detect the effect of maprotiline on the ratio of NK cells in spleen, and the maprotiline signi cantly raises the percentage of NK cells. It indicates that maprotiline might prompt the function of NK to kill the tumor cells by inhibiting the expression of PD-L1.
In this study, we con rm that maprotiline could increase the apoptosis of B16 cells, inhibit the tumor growth and improve the survival of mice burdened B16 cells. Importantly, treatment with maprotiline signi cantly inhibits the expression of PD-L1 in tumor-tissues, prompts the anti-tumor immune response of mice through increasing the ratios of immune cells in spleens and the in ltration of T cells in tumor-tissues. This study may nd a new inhibitor of PD-L1, which provides the theoretical and experimental basis to search for new drugs treated tumor in clinical.

Declarations
Ethics approval and consent to participate This article does not contain any studies with human participants. The animal studies were approved by the Ethics Committee of Xinxiang Medical University (Xinxiang, China).

Consent for publication
The authors agree to publication in the journal of Investigational New Drugs.

Availability of data and materials
The data used to support the ndings of this study are available from the corresponding author upon request. Figure 1 Effect of maprotiline on the proliferation of B16 cells in vitro. B16 cells are seeded into 96-well plates with concentration of 1.5×10 4 cells/well. 24h or 48h after being treated with maprotiline at the dose of 0, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20 and 40μg/ml, CCK-8 is added to each well and the OD value is detected according to the instruction. a Images of the representative cells in each well at 24h or 48h after being treated with maprotiline. b OD values of cells in each well at 24h or 48h after being treated with maprotiline. Data were presents as mean ± SD (n = 3). * P < 0.05 versus the Control group; # P < 0.05 versus the 20μg/ml group. Effect of maprotiline on the migration of B16 cells in vitro. B16 cells are seeded into 6-well plates with the density of 3.5×10 5 cells/well. The cell migration is detected by the method of wound healing assay. a Images of the representative cells of wound healing assay at 0h, 24h or 48h after being treated with maprotiline. b Quanti cation analysis of the nick. Data were presents as mean ± SD (n = 3). * P < 0.05 versus the Control group; # P < 0.05 versus the 5μg/ml group. Effect of treatment with maprotiline on the expression of relative proteins and cell apoptosis in vitro. B16 cells are seeded into 6-well plates with the density of 3.5×10 5 cells/well. At 24h or 48h after being treated with the maprotiline at different dose of 0, 0.625, 1.25, 2.5, 5 and 10μg/ml, the expression of relative proteins is detected by the method of western blotting and the cell apoptosis is detected by the method of Annexin V/PI analysis. a Images of the relative protein expression at 24h after being treated with maprotiline. b Images of the relative protein expression at 48h after being treated with maprotiline. c Images of cell apoptosis detected by the ow cytometry Images of the relative protein expression at 24h after being treated with maprotiline. d Images of cell apoptosis detected by the ow cytometry Images of the relative protein expression at 48h after being treated with maprotiline. * P < 0.05 versus the Control group.

Figure 4
Page 16/17 Effect of treatment with maprotiline on mice burdened B16 cells. The C57BL/6 mice are injected with 1×10 6 B16 cells and treat with PBS, 100μg maprotiline and 200μg maprotiline. The tumor weight and survival are recorded. At 7 days after the last treatment, the tumor tissues are separated for the detection of protein expression and cell apoptosis. a The procedure of therapeutic time. b Images of the representative tumor in each group. c Histogram of tumor weight in each group. d Survival rate of the mice burdened the B16 cells. e The expression of relative proteins detected by Western blotting. f Representative images of TUNEL staining in tumor-tissues. Number of mice per group are six or ten (n=6 or 10). * P < 0.05 versus the PBS group.  Effect of treatment with maprotiline on the T cell in ltration in tumor tissues. The C57BL/6 mice are injected with 1×10 6 B16 cells and treat with PBS, 100μg maprotiline and 200μg maprotiline. At day 7 th after the last treatment, the tumor tissues are separated for the detection T cell in ltration by immuno uorescence staining. a Images of CD4 + T lymphocyte in ltration in tumor tissues. b Images of CD8 + T lymphocyte in ltration in tumor tissues.